CTL Lines Research Articles

Systematic Determination of TCR–Antigen and Peptide–MHC Binding Kinetics among Field Variants of a Theileria parva Polymorphic CTL Epitope

CTLs are known to contribute to immunity toward Theileria parva, the causative agent of East Coast fever. The Tp967-75 CTL epitope from the Muguga strain of T. parva is polymorphic in other parasite strains. Identifying the amino acids important for MHC class I binding, as well as TCR recognition of epitopes, can allow the strategic selection of Ags to induce cellular immunity toward T. parva In this study, we characterized the amino acids important for MHC class I binding and TCR recognition in the Tp967-75 epitope using alanine scanning and a series of variant peptide sequences to probe these interactions. In a peptide-MHC class I binding assay, we found that the amino acids at positions 1, 2, and 3 were critical for binding to its restricting MHC class I molecule BoLA-1*023:01. With IFN-γ ELISPOT and peptide-MHC class I Tet staining assays on two parasite-specific bovine CTL lines, we showed that amino acids at positions 5-8 in the epitope were required for TCR recognition. Only two of eight naturally occurring polymorphic Tp9 epitopes were recognized by both CTLs. Finally, using a TCR avidity assay, we found that a higher TCR avidity was associated with a stronger functional response toward one of two variants recognized by the CTL. These data add to the growing knowledge on the cross-reactivity of epitope-specific CTLs and specificities that may be required in the selection of Ags in the design of a wide-spectrum vaccine for East Coast fever.

Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus

To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range. Cytotoxic effectors expressed rearranged TCR genes, perforin, granzyme, and IFN-γ. Four clonal cytotoxic lines were developed and unique TCR gene rearrangements including γδ were detected. Furthermore, catfish CTL clones were either CD4+/CD8- or CD4-/CD8-. Two CTL lines showed markedly enhanced killing of G14D-CCV targets, while the other two lines displayed a broader target range. Collectively, catfish virus-specific CTL display unique features that illustrate the diversity of the ectothermic vertebrate immune response.

In T-cell lines dually sensitized with antigens from EBV and CMV or from EBV and WT1 the T-cells specific for CMV or WT1 are often restricted by hla alleles that differ from those restricting T-cells specific for EBV

Background & Aim Virus-specific T-cells generated from transplant or 3rd party donors can induce durable remissions of post-transplant severe infections or EBV lymphomas. T-cells simultaneously sensitized with antigens from multiple viruses have also shown promise. We hypothesized that in any individual donor, immunogenic peptides from different viruses might elicit T-cell responses restricted by different HLA alleles. In HLA non-identical patients, the T-cells, specific for one virus or tumor antigen will be effective only if they are restricted by an HLA allele shared by the patient. Methods, Results & Conclusion We analyzed HLA restrictions (HLA-R) of 42 CTL lines generated by dual sensitization with either autologous EBV-transformed B-cells (BLCL) loaded with a pool of overlapping 15-mer peptides spanning the sequence of CMVpp65 (BLCL/CMV CTL) (n=20) or autologous BLCL loaded with a pool of 15-mers spanning the oncofetal protein WT-1 (BLCL/WT1 CTL) (n=22). EBV CTL lines were generated by sensitization with BLCL alone. The HLA-R of the CMVpp65-specific T-cells(CMV-T), WT1 specific T cells (WT1-T) and EBV specific T cells (EBV-T) in each T cell product were assessed by their cytotoxic activity against a panel of Cr51 labeled peptide-loaded dendritic cells and BLCL sharing a single HLA allele with the T cell donor. In 13/20 BLCL/CMV CTL(65%) and 17/22 BLCL/WT1 CTL(77%) the CMV-T or WT1-T were restricted by single HLA alleles. In 10/20(50%) BLCL/CMV CTL, CMV-T and EBV-T in the same line were restricted by the same HLA allele. However, in the other 10(50%) the CMV-T were restricted by an HLA allele different from that of the EBV-T. Similarly, in 13/22(59%) BLCL/WT1 CTL, WT1-T and EBV-T in the same line were restricted by different HLA alleles. In 2/4 BLCL/CMV CTL and 2/5 BLCL/WT1 CTL, in which the HLA-R of CMV-T or WT1-T differed from that of EBV-T in the same culture, the HLA allele differentially presenting CMV or WT1 antigen but not an EBV antigen was a prominent restricting allele of EBV-T in the EBV CTL of the same origin. This suggests that while immunodominant EBV and CMV epitopes presented by certain prevalent HLA alleles such as HLA B0702 or HLA A0201 often induce responses to both viruses, in certain dual-sensitized cultures, immunogenic epitopes from different antigens may compete for presentation by a specific HLA allele or differ in their potential to induce a dominant T-cell response.

Leukocyte immune-type receptor expressing cells increase in response to Edwardsiella ictaluri infection in Channel Catfish, Ictalurus punctatus

Abstract Unique to teleosts, leukocyte immune-type receptors (LITRs) represent a large polygenic, polymorphic family of immunoregulatory receptors. Monoclonal antibodies (mAb), CC34 and CC41, were developed against a catfish clonal CTL line and a catfish NK cell line, respectively. Both recognize members of the LITR family. One subset of LITRs, defined by reactivity with mAb CC41, is a distinct marker of a cytotoxic cell population, which expands during the antiviral response to Channel Catfish Virus (CCV). It is hypothesized that these mAb CC41-reactive LITRs function as NK-like receptors and recognize stress induced ligands on target cells. Edwardsiella ictaluri, an intracellular pathogen, primarily establishes infection in pronephros (head kidney) monocytes and may elicit a strong cytotoxic response. To explore this, small fish (<1kg) were infected with E. ictaluri via bath immersion (1×107cfu/ml). Following infection, whole blood was collected and analyzed daily by flow cytometry for mAb CC34 and CC41 staining. In parallel, an in vitro model was developed to monitor the number of CC34 and CC41-reactive cells in response to E. ictaluri infection. Briefly, head kidney monocytes and peripheral blood lymphocytes (PBL) were isolated from catfish of either susceptible or resistant background. Head kidney cells were infected with E. ictaluri (MOI 10) and then mixed with PBL from the same individual catfish and analyzed daily by flow cytometry. There was an increase in staining with both mAbs CC34 and CC41 by day 3 after infection both in vivo and in vitro. This study highlights the utilization of these mAb reagents to monitor cytotoxic cell response to E. ictaluri infection.

Loss of tapasin in human lung and colon cancer cells and escape from tumor-associated antigen-specific CTL recognition

ABSTRACTCytotoxic T-lymphocytes (CTLs) lyse target cells after recognizing the complexes of peptides and MHC class I molecules (pMHC I) on cell surfaces. Tapasin is an essential component of the peptide-loading complex (PLC) and its absence influences the surface repertoire of MHC class I peptides. In the present study, we assessed tapasin expression in 85 primary tumor lesions of non-small cell lung cancer (NSCLC) patients, demonstrating that tapasin expression positively correlated with patient survival. CD8+ T-cell infiltration of tumor lesions was synergistically observed with tapasin expression and correlated positively with survival. To establish a direct link between loss of tapasin and CTL recognition in human cancer models, we targeted the tapasin gene by CRISPR/Cas9 system and generated tapasin-deficient variants of human lung as well as colon cancer cells. We induced the CTLs recognizing endogenous tumor-associated antigens (TAA), survivin or cep55, and they responded to each tapasin-proficient wild type. In contrast, both CTL lines ignored the tapasin-deficient variants despite their antigen expression. Moreover, the adoptive transfer of the cep55-specific CTL line failed to prevent tumor growth in mice bearing the tapasin-deficient variant. Loss of tapasin most likely limited antigen processing of TAAs and led to escape from TAA-specific CTL recognition. Tapasin expression is thus a key for CTL surveillance against human cancers.

Third Party CMV-Specific Cytotoxic T Cells for Treatment of Antiviral Resistant CMV Infection after Hematopoietic Stem Cell Transplant

Adoptive immunotherapy with transplant donor derived virus specific T cells is an effective strategy for the treatment of CMV viremia and disease arising after an allogeneic hematopoietic stem cell (HSCT). This approach is not readily applicable if the donor is seronegative or not available to provide lymphocytes for in vitro expansion for CMV specific cytotoxic T lymphocytes (CMV-CTL) lines or if the CMV CTL lines derived from non-identical donors are restricted by non-shared HLA alleles. We and others have previously presented results indicating that treatment with in vitro expanded CMV-CTLs derived from an HLA partially matched third party donors can effectively treat CMV infections in the post-transplant setting. However, the patient population most likely to benefit from this approach has not been well defined.Patients at especially high risk of succumbing to CMV infection include those who acquire resistance to antiviral therapy. We now present results of treatment with banked off-the-shelf third party CMVpp65 specific CTLs in patients with genetically defined mutations in the UL54 DNA polymerase and UL97 kinase CMV genes predicting resistance to antiviral agents. Fifteen recipients of HSCT with mutations defined prior to the start of cell therapy were treated. All 15 had mutations associated with resistance to ganciclovir. Overall 5 had resistance to ganciclovir, foscarnet and cidofovir, 5 had resistance to ganciclovir and foscarnet, one had resistance to ganciclovir and cidofovir and 4 had resistance to ganciclovir alone.Third party CMV-CTLs were selected on the basis of HLA matching at high resolution at a minimum of 2/8 recipient alleles and HLA restriction of the T cells by one or more HLA alleles present in the patient. CMV-CTLS were selected from a bank of 132 lines generated under GMP conditions from normal HSCT donors specifically consented for use of their T cells in patients other than the designated transplant recipient. These 15 patients had a median age of 60.5 (7.4-70.1) years and started therapy with CMV-CTLs a median of 157 (70-564) days after reactivation of CMV. Four of these patients had CMV disease while 11 were treated for viremia alone. Prior therapy in this cohort included foscarnet (N=15) ganciclovir and/or valganciclovir (N=14) and cidofovir and/or brincidofovir (N=9). Each cycle of CMV-CTLs consisted of 3 weekly infusions of 1x106 T-cells/kg/infusion. These 15 patients received a median of 2 (1-3) cycles of CMV-CTLs. CMV-CTLs were well tolerated and there were limited toxicities. Six patients experienced AEs of which one grade 3 and one grade 4 AE were deemed possibly related to infusions with CMV-CTLs.Overall 11/15 (73.3%) patients responded to CMV-CTL therapy including 2 patients with disease with 6 CRs and 5 PRs. Overall survival at 6 months in the 11 responders and 4 non-responders was 72.7% and 25.0% respectively. Within the 6 month follow-up one of the 11 responding patients died of CMV while 3 of the 4 non-responding patients died of CMV. These results indicate that third party donor derived "off-the-shelf" CMV-CTLs can effectively treat CMV viremia or disease in patients not responding to antiviral therapy with demonstrated genetic resistance to antiviral agents. [Display omitted] DisclosuresDoubrovina:Atara Biotherapeutics: Consultancy, Research Funding. Hasan:Atara Biotherapeutics: Consultancy, Research Funding. Koehne:Atara Biotherapeutics: Consultancy.

Efficacy of Third Party BK Virus (BKV) Specific Cytotoxic T-Lymphocytes Generated By Ex Vivo Expansion for the Treatment of BKV Infection in Stem Cell Transplant Recipients, a Phase 2 Trial

Clinical manifestations of BKV hemorrhagic cystits (HC) following hematopoietic stem cell transplantation (HSCT) range from microscopic hematuria to bladder hemorrhage and renal failure. Pharmacologic treatments for HC have limited efficacy and significant adverse effects.Here, we report the preliminary analysis of adoptive immunotherapy with most closely HLA-matched third party BKV-specific CTL lines (BKV-CTLs) generated by ex vivo expansion.We generated a bank of BKV-specific CTLs from 17 virus-immune healthy donors (mean age, 55; range, 25 to 75 years). Mononuclear cells enriched from donor buffy coats were stimulated with BKV peptide mix from capsid proteins VP1, VP2, VP3, large T antigen (LT) and small T antigen (ST) in the presence of IL-2, IL-7 and IL-15 for 14 days. At the end of culture, the cells were harvested and cryopreserved until use.Patients with symptomatic BKV cystitis after HSCT for leukemia or lymphoma were eligible. Patients with acute GVHD grades II-IV, those receiving >0.5 mg/kg systemic steroids/day or on treatment with cidofovir or leflunomide were excluded. A search for the most closely HLA-matched donor was initiated through the MDACC cell therapy donor bank. BKV-specific T cells were infused intravenously directly after thawing. Patients could receive up to 2 infusions.BKV PCR and clinical symptoms were monitored every week for 28 days after infusion. Complete response was defined as complete resolution of symptoms and gross hematuria and partial response (PR) as decrease in grade of HC from 3 (urinary blood clots) or 4 (red cell transfusion requirement/renal impairment) to 2 (macroscopic hematuria) or 3 respectively. Patients with stable disease (SD) or progression had insufficient changes to qualify as PR or had an increase in symptoms or worsening hematuria, respectively.To date, 10 patients (2 HLA-matched related, 5 HLA-matched unrelated and 3 haploidentical HSCT recipients) have received BKV-CTLs for BK cystitis. The median number of BKV-specific CD3+IFN-gamma+ and CD3+IL-2+ T-cells infused was 10e3/kg and 4.8e3/kg, respectively. The ratio of BKV-specific CD4+/CD8+ T cells in the infused product was 7.8 (range 3.1-22.1). There were no infusion-related adverse effects. Based on end-organ response, BKV-CTLs controlled infection in 9/9 evaluable patients with BKV cystitis (4 CRs and 5 PRs)- one patient was not evaluable due to disease relapse, sepsis and anuria. All patients achieved a response by day 14 post-infusion. Two of 5 patients with PR received a second BK CTL infusion from a different donor. One had an initial PR to the first CTL infusion but symptoms progressed after 6 weeks. A second infusion resulted in a PR with reduction in BKV PCR and symptoms. The other patient achieved CR within 28 days of the second infusion. The time to second infusion was 6 and 3 weeks, respectively. The response in all patients was sustained.Following infusion, we detected high frequencies of polyfunctional BKV-specific CD4+ and CD8+ T-cells, capable of producing IL-2, IFN-gamma and TNF-alpha in response to ex vivo stimulation with BKV peptide pools (VP1, VP2, VP3, LT and ST). The response was mainly CD4+ T cell dominant, reflecting the composition of the infused BKV T cell line and consistent with previous reports of a predominantly CD4-mediated T cell response in BKV infection.De novo grade 2-4 acute graft versus host disease (aGVHD) occurred in 1/10 patients. The patient developed grade 2 duodenal GVHD 14 days following infusion. A second patient had recurrence of a prior skin GVHD and developed grade 2 aGVHD 9 days after infusion. Both were successfully treated with 1mg/kg/day systemic steroids.This analysis demonstrates the safety, feasibility and efficacy of administration of most closely HLA-matched BKV CTLs for the treatment of BKV cystitis in HSCT patients. The response rate of 100% in the first 10 patients treated with BKV CTLs is promising. DisclosuresCiurea:Cyto-Sen Therapeutics: Equity Ownership; Spectrum Pharmaceuticals: Other: Advisory Board.

Non-cognate bystander cytolysis by clonal epitope-specific CTL lines through CD28–CD80 interaction inhibits antibody production: A potential caveat to CD8 T-cell immunotherapy

Adoptive transfer of virus epitope-specific CD8 T cells is an immunotherapy option to control cytomegalovirus (CMV) infection and prevent CMV organ disease in immunocompromised solid organ transplantation (SOT) and hematopoietic cell transplantation (HCT) recipients. The therapy aims at an early, selective recognition and cytolysis of infected cells for preventing viral spread in tissues with no adverse immunopathogenic side-effects by attack of uninfected bystander cells. Here we describe that virus epitope-specific, cloned T-cell lines lyse target cells that present the cognate antigenic peptide to the TCR, but simultaneously have the potential to lyse uninfected cells expressing the CD28 ligand CD80 (B7-1). While TCR-mediated cytolysis requires co-receptor CD8 and depends on perforin, the TCR-independent and viral epitope-independent cytolysis through CD28–CD80 signaling does not require CD8 on the effector cells and is perforin-independent. Importantly, this non-cognate cytolysis pathway leads to bystander cytolysis of CD80-expressing B-cell blasts and thereby inhibits pan-specific antibody production.

NFATc2 is an intrinsic regulator of melanoma dedifferentiation.

Melanoma dedifferentiation, characterized by the loss of MITF and MITF regulated genes and by upregulation of stemness markers as CD271, is implicated in resistance to chemotherapy, target therapy and immunotherapy. The identification of intrinsic mechanisms fostering melanoma dedifferentiation may provide actionable therapeutic targets to improve current treatments. Here, we identify NFATc2 transcription factor as an intrinsic regulator of human melanoma dedifferentiation. In panels of melanoma cell lines, NFATc2 expression correlated inversely with MITF at both mRNA and protein levels. NFATc2(+/Hi) melanoma cell lines were CD271(+) and deficient for expression of melanocyte differentiation antigens (MDAs) MART-1, gp100, tyrosinase and of GPNMB, PGC1-α and Rab27a, all regulated by MITF. Targeting of NFATc2 by small interfering RNA, short hairpin RNA and by an NFATc2 inhibitor upregulated MITF, MDAs, GPNMB, PGC-1α, tyrosinase activity and pigmentation and suppressed CD271. Mechanistically, we found that NFATc2 controls melanoma dedifferentiation by inducing expression in neoplastic cells of membrane-bound tumor necrosis factor-α (mTNF-α) and that melanoma-expressed TNF-α regulates a c-myc-Brn2 axis. Specifically, NFATc2, mTNF-α and expression of TNF receptors were significantly correlated in panels of cell lines. NFATc2 silencing suppressed TNF-α expression, and neutralization of melanoma-expressed TNF-α promoted melanoma differentiation. Moreover, silencing of NFATc2 and TNF-α neutralization downmodulated c-myc and POU3F2/Brn2. Brn2 was strongly expressed in NFATc2(+/Hi) MITF(Lo) cell lines and its silencing upregulated MITF. Targeting of c-myc, by silencing or by a c-myc inhibitor, suppressed Brn2 and upregulated MITF and MART-1 in melanoma cells. The relevance of NFATc2-dependent melanoma dedifferentiation for immune escape was shown by cytolytic T-cell assays. NFATc2(Hi) MITF(Lo) MDA(Lo) HLA-A2.1(+) melanoma cells were poorly recognized by MDA-specific and HLA-A2-restricted CTL lines, but NFATc2 targeting significantly increased CTL-mediated tumor recognition. Taken together, these results suggest that the expression of NFATc2 promotes melanoma dedifferentiation and immune escape.

Administration of LMP-specific cytotoxic T-lymphocytes to patients with relapsed EBV-positive lymphoma post allogeneic stem cell transplant

EBV associated tumors in the immunocompetent host express the type II latency antigens LMP1 and LMP2 which can serve as potential targets for immunotherapy. For many patients with Chronic Active Epstein Barr Virus (CAEBV) infection and relapsed Hodgkin’s Lymphoma (HL), the only potential curative option is allogeneic HSCT. However, relapse rates remain high (>50%). We hypothesized that the administration of donor-derived LMP-CTL would rapidly restore LMP-specific T-cell immunity and prevent relapse in these high-risk patients. LMP-CTL lines were expanded using donor-derived monocytes and EBV transformed lymphoblastoid cell lines, transduced with an adenoviral vector expressing either LMP2 (n=4) or ΔLMP1 and LMP2 (n=15). Characterization of CTL lines at cryopreservation revealed CD4+ and CD8+ cells which were predominantly effector and effector memory phenotype. CTL lines had LMP2 (+/-LMP1) specific activity as determined by ELISPOT and cytotoxicity assays. Sixteen patients (n=6 HL, n=4 B-cell CAEBV/NHL, n=6 T-cell CAEBV/NHL) received CTL (dose range: 0.4-3x10e8/m2) approximately 100 days post HSCT. No toxicities related to CTL were observed. Fourteen patients were in remission at the time of CTL infusion and 13/14 of these patients remained in remission for a median of 30 months post CTL (range 3-90 months). One patient relapsed 6 months post CTL and died of progressive disease. Two patients received CTL for relapsed disease after HSCT and both died of disease progression. In general, donor-derived LMP-CTL seem to be well tolerated in high-risk patients with EBV+ lymphoma or CAEBV post HSCT. In addition, the use of donor-derived tumor-directed T-cells as adjuvant therapy post HSCT may restore LMP-specific T-cell immunity and prevent relapse in this high-risk patient population. Moreover, we are able to use these normal donor–derived LMP-CTL to develop a bank of LMP-specific CTL for third party use as an “off the shelf” therapeutic for EBV associated malignancies.

The Src tyrosine kinase Lck binds to CD2, CD4-1, and CD4-2 T cell co-receptors in channel catfish, Ictalurus punctatus

The binding of the lymphocyte specific protein tyrosine kinase (Lck) to T cell co-receptors is required for T cell development and activation. In mammals, Lck initiates signal transduction by binding to CD4 and CD8 co-receptors and phosphorylating ITAMs in the cytoplasmic tail of the CD3 molecules and the ζ chains. In addition, Lck can also bind to the adhesion molecule CD2 and trigger T cell activation. In this study, Lck and CD2 homologs were identified and characterized in channel catfish, Ictalurus punctatus. Lck and CD2 mRNAs were specifically expressed by clonal T cell lines, including both CD4+ and CD4−CD8− CTL lines, and in mixed lymphocyte cultures (MLC). Western blot analyses using anti-trout Lck and anti-human p-Lck antibodies demonstrated that Lck protein is expressed in catfish clonal CTL and is phosphorylated at a conserved tyrosine residue. Because of the lack of CD8+ CTL lines as well as the absence of CD8 message in MLC, we performed magnetic bead binding assays to correlate CD2, CD4, and CD8 co-receptor expression with Lck binding ability. Recombinant Lck reproducibly bound to CD2, CD4-1, and CD4-2, but not to CD8α or CD8β. These data provide one possible explanation for the apparent low numbers of CD8+ CTL and the presence of CD4+ and CD4−CD8−CD2+ CTL in catfish.

Selection of Thymocytes Expressing Transgenic TCR Specific for a Minor Histocompatibility Antigen, H60.

Minor histocompatibility antigens are MHC-bound peptides and contribute to the generation of allo-responses after allogeneic transplantation. H60 is a dominant minor H antigen that induces a strong CD8 T-cell response in MHC-matched allogeneic transplantation settings. Here, we report establishment of a TCR transgenic mouse line named J15, wherein T cells express TCRs specific for H60 in complex with H-2Kb, and different fates of the thymocytes expressing J15 TCRs in various thymic antigenic environments. Thymocytes expressing the J15 TCRs were positively selected and differentiated into CD8+ single positive (SP) cells in the thymus of C57BL/6 mice, wherein the cognate antigen H60 is not expressed. However, thymocytes were negatively selected in thymus tissue where H60 was transgenically expressed under the control of the actin promoter, with double-positive stages of cells being deleted. Despite the ability of the H60H peptide (LTFHYRNL) variant to induce cytotoxic activity from H60-specific CTL lines at ~50% of the activity induced by normal H60 peptides (LTFNYRNL), J15-expressing thymocytes were positively selected in the thymus where the variant H60H was transgenically expressed. These results demonstrate that a single amino-acid change in the H60 epitope peptide influences the fate of thymocytes expressing the cognate TCR.

Banked, GMP Grade Third Party T-Cell Lines Specific for CMVpp65 Epitopes Presented By Certain Prevalent HLA Alleles More Consistently Clear CMV Infections in a Genetically Heterogeneous Population of HSCT Recipients

Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HSCT donors or HLA partially matched third party donors can eradicate EBV, CMV or adenovirus infections in a high proportion of allogeneic HSCT recipients. At our center, the response rate to third party CMVpp65-specific T-cells (CMVCTLs) has been about 60%. The specific characteristics of CTLs from third party donors that can predict efficacy after adoptive transfer have not been fully defined. However, current evidence indicates that for third party CTLs to be effective, CTL lines selected for treatment must at least be restricted by an HLA allele shared by the cells infected in the patient and the T-cell donor. We have developed a CMV CTL bank containing 132 CTL lines that was generated using a pool of overlapping peptides spanning the sequence of CMVpp65. Each CMV CTL line in the bank has been characterized as to T-cell memory phenotype, cytokine profile, epitope specificity and the HLA allele restriction of the cytotoxic T-cells. Although our pool of CTL donors has inherited a diverse group of widely prevalent HLA alleles, the cytotoxic activity of the T-cell lines in the bank is restricted by only 11 alleles. Nevertheless, these T-cells provided an appropriately restricted CTL line for 51 of 56 requested cases. In the present study, we examined 52 HSCT recipients who had received CMV CTLs of defined HLA restriction from either their HSCT donor (n=23) or a third party donor (n= 29) to determine whether and to what degree specific immunodominant T-cells restricted by specific HLA alleles exhibited differential clinical activity following adoptive transfer. We did this analysis because in analyzing responses to epitopes presented by these alleles, we noted that epitopes presented by certain alleles elicited quantitatively greater in-vitro responses as measured by the number of IFNγ+ or tetramer+ T-cells/106T-cells administered.For 9 CTL lines used in our series, the epitope eliciting the immunodominant T-cell response was an 11-15 aa peptide that was presented by both an HLA class-I and class-II allele. These class-I and class-II shared epitopes elicited more robust, multifunctional CD8+ and CD4+ T-cells that generated IFNγ, TNFα and IL-2. 8 of 9 patients treated with one of these CTL lines rapidly cleared viremia and end organ disease.Another group of 4 CTL lines responded to 2 epitopes, 1 dominant and 1 subdominant, that were restricted by one HLA A and one B allele; specifically A01 and B08, A 01 and B35 and A24 and B35. Treatment with such dual epitope responsive CTL lines also yielded complete responses in 4/4 cases.Among lines specific for single immunodominant epitopes, HLA restricted CMV CTLs responding to 4 CMVpp65 epitopes also consistently induced rapid clearance of viremia and organ disease including colitis, retinitis and encephalitis; NLVPMVATV (14/16 Complete responses CR), TPRVTGGGAM/RPHERNGFTV (7/7 CR) and HERNGFTV (6/6 CR) presented by HLA A0201, B0702 and allelic variants of B40 or B44 (B4001-06 and/B4401-03 ) respectively.In contrast, none of the 10 patients treated CTL lines responding to epitopes presented by HLA A2601, A2407, A2902, B0705, B5001, or allelic variants of B35 responded to CTL treatment and progressed.These findings suggest a hierarchy of epitopes presented by prevalent HLA alleles that may exhibit more consistent clinical activity in-vivo. Selection strategies utilizing such data may permit the development of a CTL selection algorithm for CMV CTLs derived from third party donors for treatment of CMV infections that would, more consistenly, induce clearance of CMV viremia and disease. These data also offer the potential to focus strategies for generation of CMV CTLs using a limited peptide pool or a panel of AAPCs expressing specific HLA alleles, thereby making this treatment approach more financially and ligisticaly feasible. These data could also be applied toward the development of an effective CMV vaccine for high risk individuals. DisclosuresNo relevant conflicts of interest to declare.

Graft Versus Leukemia Response without Graft Versus Host Disease Elicited By Adoptively Transferred Multivirus-Specific T-Cells

Refractory acute lymphoblastic leukemia (ALL) remains a disease with poor prognosis, and a small percentage of patients that undergo allogeneic stem cell transplantation will respond. Following allogeneic transplant, donor T-cells may cause graft-versus-host (GVH) alloreactions but also confer immunity to viruses and exert a graft-versus-leukemia (GVL) effect contributing to maintenance of minimal residual disease and cure of leukemia. Control of viral disease can be enhanced by infusion of ex-vivo expanded virus-specific T-cells. Since some malignancies such as Epstein-Barr virus (EBV)-driven lymphoproliferative diseases can express viral antigens they can be successfully eradicated by EBV specific CTL. However cross-reactivity between virus-specific T cells and non-viral hematological malignancies has not been described.Here we report for the first time an apparent GVL effect against ALL from ex-vivo expanded multivirus specific CTL. A 12-year-old boy with refractory ALL received a haploidentical transplant from his mother. As prophylaxis for EBV, CMV and adenovirus, he received ex-vivo expanded virus-specific donor T-cells 3.5 months after HSCT. Four weeks later leukemic blasts bearing the E2A deletion, appeared transiently in the blood followed by a FISH negative hematological remission, which was sustained until a testicular relapse 3.5 months later. The infused T cell line displayed predominant reactivity against CMV antigens and epitope mapping indicated broad reactivity with a number of pp65-derived epitopes including 1.88% of CD8+ T-cells that recognized the HLA-A24-restricted pp65 epitope QYDPVAALF. Within 2weeks post-CTL, markedly increased frequencies of CMV and adenovirus-specific T-cells were detectable in the patient suggesting that the virus specific T-cells expanded in-vivo. The occurrence of leukemic relapse followed by remission at the peak of virus-specific immune reconstitution suggested that virus-specific T cells were reactive against host leukemia. The observation conformed more with a GVL rather than a GVH effect since the patient did not develop GVHD following the T-cell infusion and no anti-recipient CTL activity was demonstrated. To explore leukemia-specific targeting by the virus-specific CTL, the T cell line was incubated with patient PHA blasts, patient ALL blasts and with donor EBV-LCL loaded with the CMV pp65 peptide library. CD4+ T cells, and, to a lesser extent CD8+ T cells, displayed a strong, polyfunctional reactivity against patient leukemia blasts, indicating that the virus-specific T cells were cross-reactive with the leukemia cells. (Figure 1a and 1b) Additionally, using IFN-γ ELISpot, the virus-specific T cells not only recognized viral-derived overlapping peptides, but also peptides derived from the tumor- associated antigens (TAA), Wilm’s tumor-1 (WT-1), preferentially expressed antigen in melanoma (PRAME), and melanoma-associated antigen-3 (MAGEA3) known to be expressed at high frequencies on ALL blasts. (Figure 2) The possibility that the effect was mediated by anti-leukemic NK cells was eliminated by gating CD4+ and CD8+ T-cell subpopulations from the CTL line, separately. Despite a temporary remission, our patient eventually relapsed in the testis, recognized as an immune privileged leukemia sanctuary site and at a time when CMV, EBV or AdV specific T cells were no longer detectable in the peripheral blood. Evidence for a role of virus reactivation in reducing leukemia relapses post allogeneic HSCT has accumulated. This is, to our knowledge, the first report of virus-specific T cells with heterologous reactivity against ALL cells eliciting a GVL response without GVHD suggesting the need for further studies to identify how frequently virus-specific CTL lines can recognize and kill leukemia. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Third Party Donor Derived CMV Specific T Cells for the Treatment of Refractory CMV Viremia and Disease after Hematopoietic Stem Cell Transplant

▪Adoptive immunotherapy with transplant donor derived virus specific T cells is an effective strategy for the treatment of CMV viremia and disease arising after an allogeneic hematopoietic stem cell (HSCT). This approach is not readily applicable if the donor is seronegative or not available to provide lymphocytes for in vitro expansion for CMV specific cytotoxic T lymphocytes (CMV-CTL) lines or if the CMV CTL lines derived from non-identical donors are restricted by non-shared HLA alleles.To date, we have treated 38 consecutive patients with overt CMV disease (N=12) or CMV viremia (N=26) persisting despite >2 weeks of antiviral drugs with in vitroexpanded CMV-CTLs derived from an HLA partially matched third party donor. CMV CTLS were selected from a bank of 132 lines generated under GMP conditions from normal HSCT donors specifically consented for use of their T cells in patients other than their designated transplant recipient. Patients were recipients of unmodified (n=6), T cell depleted (n=27) or unrelated cord blood (n=5) HSCT.Third party CMV-CTLs were selected on the basis of HLA matching at a minimum of 2/8 recipient alleles and HLA restriction of the T cells by one or more HLA alleles present in the patient. A total of 24 distinct CMV-CTL lines were used. The HLA restriction of the CMV CTLs was at a single HLA allele (n=19), at two alleles (n=4) and at >than two alleles (n=1). Patients received infusions of 3rd party CMV-CTLs after failing at least 14 days, and a median of 113 (26-402) days of prior therapy with a median of 4 anti-viral agents. Patients received 3 weekly infusions most at 1x106CMV-CTL/kg/infusion. Four patients were evaluated only for toxicity from CMV-CTLs based on changes in their concomitant anti-viral therapy close to the time of CMV-CTL therapy. Responses in patients treated for viremia alone were considered complete if the viremia resolved completely and partial if it fell by 2 logs. Responses in disease were considered complete (CR) if all detectable CMV viremia and disease resolved and partial (PR) if patients became asymptomatic.Of the 25 evaluable patients with viremia alone 5 achieved CR, and 9 PR. In 5 CMV viremia was reduced by less than 2 logs, and 6 had progressive viremia or developed disease. Of the 8 evaluable patients treated for disease 2 achieved a CR, 3 a PR, one had stable disease and two progressed. A total of nine patients ultimately died of CMV. There were limited toxicities associated with CMV CTL infusions. Two patients developed progression of presumed CMV pneumonitis. No patients developed de novo GvHD or a flare of prior GvHD in association with mis-matched third party CMV CTLs.This study demonstrates a high response rate among patients with otherwise refractory CMV viremia and disease. CMV CTLs can be effective when selected based on restriction to shared alleles despite significant HLA disparity. The bank of CMV specific T cells can provide an immediate source of HLA partially matched appropriately restricted T cells for adoptive immunotherapy to treat CMV viremia and disease including disease isolated to the CNS. This enables treatment early in the course of disease and the use of CMV-CTL lines previously prepared and characterized in terms of HLA restriction. This is anticipated to maximize the response rate as well as minimize toxicity from anti-viral therapy. DisclosuresOff Label Use: Clofarabine off label for bone marrow transplant. Melphalan off label for bone marrow transplant. Thiotepa off label for bone marrow transplant..

Dual-Specificity CAR+ T Cells to Target B-Cell Malignancies and Opportunistic Fungal Infection

s / Biol Blood Marrow Transplant 20 (2014) S128eS150 S132 CTL was characterized as to eps specificity and restricting HLA allele. The pool of CTL donors inherited HLA alleles in frequencies similar to those in caucasian and black populations, with certain alleles overrepresented. In 55% of the CTL lines, the ID T-cell response (TCr) was restricted (restr.) by 3 HLA alleles: A0201(25%), B0702(21%) and B 3501-11(9%); 45% of CTL lines were restr. by other class-I alleles, and 17/123(14%) were restr. by class-II alleles. Certain CMVpp65 15 mers contained overlapping eps presented by both class-I and class-II, which elicitedmore robust TCr. CTLs from all B0702+ donors (26/26) were restr. by B0702. In comparison, CTLs from 30/39(77%) A0201+ donors and 9/19 (47%) HLA B3501-11+ donors were restr. by A0201 and B3501-11 respectively. Donors coinheriting A0201 and B 0702 (9/9) universally demonstrated B0702 restr. ID TCr. In comparison, 11/12 (91.6%) donors coinheriting A0201 and B 4401-04 demonstrated HLA A0201 restr. CTLs. Strikingly, only 1/123 and 0/123 A1101+ and A0301+ donors respectively demonstrated CTLs restr. by these commonly inherited class eI alleles. This analysis thus indicates an immunodominance hierarchy for CMVpp65 eps and their presenting HLA alleles. In a series of 239 MUD, 137 MMUD, and 100 DUCB consecutive HSCT at MSKCC, we could identify an immediately available CMVpp65 specific CTL line matched with the patient at 2-3 alleles and restr. by a shared HLA allele in this GMP-CTL bank in 86%, 89% and 80% cases respectively. This CTL bank thus provides a clinical reagent for the treatment of CMV infections in HSCT recipients. The delineated immunodominance hierarchy for CMVpp65 may also facilitate selection of an appropriately restr. CTL line for treatment with predicted activity in the recipient. We can also successfully generate CMV-CTLs against subdominant CMVpp65 eps presented prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs). Expansion of this bank with CTLs generated with AAPCs can broaden its applicability to almost all HSCT recipients.

Generation and Characterization of a Third Party GMP Grade Bank of CMV Specific T-Cells for Adoptive Immunotherapy of CMV Infections in Recipients of HSCT from Cord Blood or Seronegative Donors

Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HLA partially matched third party donors can also be effective in a proportion of patients developing EBV lymphomas, or infections due to CMV or adenovirus following transplants from seronegative donors. Such third party donor derived CTLs offer an off the shelf reagent for treatment of viral infections developing after transplant. However, the immunodominant cytotoxic activity exhibited by the CTLs is directed against specific epitopes of the viral protein and restricted by 1-2 HLA alleles. Therefore, it is critical that the T-cells administered from third party donors can recognize viral epitopes presented on shared HLA alleles. We have established a bank of 119 CMV specific T-cell lines (CMV CTLs) generated using autologous APCs loaded with a pool of overlapping peptides spanning the sequence of the dominant immunogenic protein CMVpp65. Each of these 119 CMV CTL lines has been characterized as to the epitope inducing T-cell response as well as the HLA allele restricting the epitope specific T-cell response. Epitopes were identified using an overlapping grid of peptide pools and the HLA restriction by cytotoxic activity against peptide loaded EBVBLCLs matched at a single HLA allele with the T-cell donor. The distribution of the common HLA alleles among the donors for these CTL lines was predominantly within the distribution of HLA allele frequencies represented in the caucasian and black populations, except for HLA A0201 and B0702, which were over represented ( 33% vs 25% and 21% vs 8.7% respectively). In 54% of the CTL lines, the immunodominant T-cell responses were restricted by HLA A0201 (25%), B0702 (21%) and B 3501-11(8%), and in the remaining 50%, the responses were restricted by other HLA class-I alleles, while only 16/119 lines (13%) were restricted by HLA class-II alleles. All 25 donors inheriting HLA B0702 (25/25) demonstrated HLA B0702 restricted CMV CTL responses, while 30/39 (77%) donors inheriting HLA A0201 and 9/19 (47%) donors inheriting HLA B3501-11 demonstrated HLA A0201 and B3501-11 restricted CMV CTL responses. Among all 9 donors co-inheriting HLA A0201 and B 0702, the immunodominant T-cell response was restricted by B0702. Among 12 donors co-inheriting A0201 and B 4401-04, 11/12 (91.6%) demonstrated immunodominant CMV CTL responses restricted by A0201; 1 donor also co-inherited HLA B0702 whose response was restricted by B0702. Therefore, an immunodominance hierarchy for HLA class-I alleles presenting the dominant CMVpp65 epitope was evident through this analysis among these 119 donors and was as follows: B 0702, A0201, B3501-11, A2601, B44, B40, B4201, A0101, B 1801. Strikingly, only 1 of 119 donors demonstrated T-cell responses restricted by A1101; a commonly inherited HLA class –I allele. In a series of 239 consecutive HLA matched related or unrelated transplants (MUD) and 137 HLA mismatched unrelated (MMUD) transplants, and 100 cord blood transplants conducted at our center, in 86%, 89% and 80% of the cases respectively, we could identify a CMV CTL line restricted by a shared HLA allele and matched at 2-3 alleles within this GMP grade CTL bank that would be immediately available for treatment of CMV infection. Appropriately restricted CMV CTLs would only be available in 60-70% of MMUD transplant and none of the cord blood transplants without this approach. This CMV CTL bank therefore represents a readily available clinical reagent for the treatment of resistant CMV infections developing in post transplant patients. The characterization of the CTLs has also enabled the further elucidation of immunodominant CMVpp65 epitopes and hierarchies. Since we have previously shown that CMV CTLs can be generated against subdominant epitopes presented by both common HLA alleles as well as less prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs), expansion of this bank using T-cell sensitized against CMVpp65 presented on such AAPCs should broaden the applicability of this bank to all HSCT recipients. Disclosures: No relevant conflicts of interest to declare.

Perforin Expression in <i>Theileria parva</i> Specific Cytotoxic T Cells Correlates with Cytotoxicity

Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.

Interaction of CD99 with Its Paralog CD99L2 Positively Regulates CD99L2 Trafficking to Cell Surfaces

Mouse CD99 and its paralog CD99-like 2 (CD99L2) are surface proteins implicated in cellular adhesion and migration. Although their distributions overlap in a wide variety of cells, their physical/functional relationship is currently unknown. In this study, we show the interaction between the two molecules and its consequence for membrane trafficking of mouse (m)CD99L2. The interaction was analyzed by bimolecular fluorescence complementation, immunoprecipitation, and fluorescence resonance energy transfer assays. When coexpressed, mCD99 formed heterodimers with mCD99L2, as well as homodimers, and the heterodimers were localized more efficiently at the plasma membrane than were the homodimers. Their interaction was cytoplasmic domain-dependent and enhanced mCD99L2 trafficking to the plasma membrane regardless of whether it was transiently overexpressed or endogenously expressed. Surface levels of endogenous mCD99L2 were markedly low on thymocytes, splenic leukocytes, and CTL lines derived from CD99-deficient mice. Importantly, the surface levels of mCD99L2 on mCD99-deficient cells recovered significantly when wild-type mCD99 was exogenously introduced, but they remained low when a cytoplasmic domain mutant of mCD99 was introduced. Our results demonstrate a novel role for mCD99 in membrane trafficking of mCD99L2, providing useful insights into controlling transendothelial migration of leukocytes.

Generation and Characterization Of a Third Party GMP Grade Bank Of CMV Specific T-Cells For Adoptive Immunotherapy Of CMV Infections In Recipients Of HSCT From Cord Blood Or Seronegative Donors

Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HLA partially matched third party donors can also be effective in a proportion of patients developing EBV lymphomas, or infections due to CMV or adenovirus following transplants from seronegative donors. Such third party donor derived CTLs offer an off the shelf reagent for treatment of viral infections developing after transplant. However, the immunodominant cytotoxic activity exhibited by the CTLs is directed against specific epitopes of the viral protein and restricted by 1-2 HLA alleles. Therefore, it is critical that the T-cells administered from third party donors can recognize viral epitopes presented on shared HLA alleles. We have established a bank of 119 CMV specific T-cell lines (CMV CTLs) generated using autologous APCs loaded with a pool of overlapping peptides spanning the sequence of the dominant immunogenic protein CMVpp65. Each of these 119 CMV CTL lines has been characterized as to the epitope inducing T-cell response as well as the HLA allele restricting the epitope specific T-cell response. Epitopes were identified using an overlapping grid of peptide pools and the HLA restriction by cytotoxic activity against peptide loaded EBVBLCLs matched at a single HLA allele with the T-cell donor.The distribution of the common HLA alleles among the donors for these CTL lines was predominantly within the distribution of HLA allele frequencies represented in the caucasian and black populations, except for HLA A0201 and B0702, which were over represented ( 33% vs 25% and 21% vs 8.7% respectively). In 54% of the CTL lines, the immunodominant T-cell responses were restricted by HLA A0201 (25%), B0702 (21%) and B 3501-11(8%), and in the remaining 50%, the responses were restricted by other HLA class-I alleles, while only 16/119 lines (13%) were restricted by HLA class-II alleles.All 25 donors inheriting HLA B0702 (25/25) demonstrated HLA B0702 restricted CMV CTL responses, while 30/39 (77%) donors inheriting HLA A0201 and 9/19 (47%) donors inheriting HLA B3501-11 demonstrated HLA A0201 and B3501-11 restricted CMV CTL responses. Among all 9 donors co-inheriting HLA A0201 and B 0702, the immunodominant T-cell response was restricted by B0702. Among 12 donors co-inheriting A0201 and B 4401-04, 11/12 (91.6%) demonstrated immunodominant CMV CTL responses restricted by A0201; 1 donor also co-inherited HLA B0702 whose response was restricted by B0702. Therefore, an immunodominance hierarchy for HLA class-I alleles presenting the dominant CMVpp65 epitope was evident through this analysis among these 119 donors and was as follows: B 0702, A0201, B3501-11, A2601, B44, B40, B4201, A0101, B 1801. Strikingly, only 1 of 119 donors demonstrated T-cell responses restricted by A1101; a commonly inherited HLA class –I allele.In a series of 239 consecutive HLA matched related or unrelated transplants (MUD) and 137 HLA mismatched unrelated (MMUD) transplants, and 100 cord blood transplants conducted at our center, in 86%, 89% and 80% of the cases respectively, we could identify a CMV CTL line restricted by a shared HLA allele and matched at 2-3 alleles within this GMP grade CTL bank that would be immediately available for treatment of CMV infection. Appropriately restricted CMV CTLs would only be available in 60-70% of MMUD transplant and none of the cord blood transplants without this approach. This CMV CTL bank therefore represents a readily available clinical reagent for the treatment of resistant CMV infections developing in post transplant patients. The characterization of the CTLs has also enabled the further elucidation of immunodominant CMVpp65 epitopes and hierarchies. Since we have previously shown that CMV CTLs can be generated against subdominant epitopes presented by both common HLA alleles as well as less prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs), expansion of this bank using T-cell sensitized against CMVpp65 presented on such AAPCs should broaden the applicability of this bank to all HSCT recipients. Disclosures:No relevant conflicts of interest to declare.